LOCUS       sequence-193270-        6475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mouse CB2 cannabinoid receptors and eGFP in the 
            presence of Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6475)
  AUTHORS   Li Y, Kim J
  TITLE     Distinct roles of neuronal and microglial CB2 cannabinoid receptors 
            in the mouse hippocampus.
  JOURNAL   Neuroscience. 2017 Sep 6. pii: S0306-4522(17)30629-2. doi: 
            10.1016/j.neuroscience.2017.08.053.
  PUBMED    28888955
REFERENCE   2  (bases 1 to 6475)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Neuroscience. 2017 Sep 6. pii: S0306-4522(17)30629-2. doi: 
            10.1016/j.neuroscience.2017.08.053."
COMMENT     Sequence Label: pAAV-CBh-DIO-CB2-EGFP
FEATURES             Location/Qualifiers
     source          1..6475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        145..430
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        432..709
                     /label=chicken beta-actin promoter
     intron          710..938
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     protein_bind    complement(968..1001)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1052..1085)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1222..1935,1936..1938,1939..1941))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1225..1935,1936..1938,1939..1941))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1267..1288)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1616..1635
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1875..1896
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1960..2016)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(2026..2049)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    3143..3176
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3227..3260
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3330..3350)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3738..3878
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3738..3867
                     /label=AAV2 ITR
     rep_origin      3953..4408
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4040..4059)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4250..4271
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4425..4444)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4544..4566
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4604..4622)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4690..4794
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4795..4863,4864..5655)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5013..5032)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5826..6414
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6315..6334
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtcgttac ataacttacg gtaaatggcc cgcctggctg
      181 accgcccaac gacccccgcc cattgacgtc aatagtaacg ccaataggga ctttccattg
      241 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      301 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattgtgc
      361 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      421 tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
      481 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg
      541 gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag
      601 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
      661 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
      721 gacgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct
      781 gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta
      841 attagctgag caagaggtaa gggtttaagg gatggttggt tggtggggta ttaatgttta
      901 attacctgga gcacctgcct gaaatcactt tttttcaggt tggggatcct ctagagtcga
      961 ctccggaata acttcgtata ggatacttta tacgaagtta tgcagaatgg tagctggatt
     1021 gtagctgcta ttagcaatat gaaacctctt aataacttcg tatagcatac attatacgaa
     1081 gttatggcgc gccttatcta gatttagcca ccttacttgt acagctcgtc catgcncgak
     1141 wrksawymyg rmrsckstta mtaacttcrk mwagcataca ttatacgaag ttatggcgcg
     1201 ccttatctag atttagccac cttacttgta cagctcgtcc atgccgagag tgatcccggc
     1261 ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag
     1321 ggcggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg
     1381 ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat
     1441 cttgaagttc accttgatgc cgttcttctg cttgtcggcc atgatataga cgttgtggct
     1501 gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc
     1561 cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt
     1621 gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc
     1681 ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc
     1741 gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat
     1801 gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa
     1861 cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag
     1921 ctcctcgccc ttgctcacca tggtgccggt agcgctagca ggtccagggt tctcctccac
     1981 gtctccagcc tgcttcagca ggctgaagtt agtagctccg cttcccttgt catcgtcatc
     2041 cttgtaatcc tcgagcggcc gggtacgcgt ggtggttttc acatcagcct ctgtttctgt
     2101 aaccgaggac cttgggcctt cttctttccc ctcaggtccg aggccctgta gatacttctt
     2161 ccagcctatc aggcagtgct gggcagcaga gcgaatctct ccactccgca gggcgtaaat
     2221 gataggattg accatagagt taacaaggca cagcatggaa cagaaggcga aggcctcctt
     2281 gacctggtca ctcagcgtgg tgaccaggct gtggcccatg agagccagtg cagggaacca
     2341 gcatatgagc agcacagcca gcaccaggcc cagagtcttg gccaacctca cgtctagccg
     2401 catccgagct atcccaggca cctgcctgtc ctggtgctca gccaaggtgg ctacatgccg
     2461 gtgggctttc cagaggacat acccataggt atagatgatg ccggaaaaga ggatggcaat
     2521 gaatagaagc cagcccagta ggtagtcgtt agggatcagt gggaaaagct cagagcaggg
     2581 actagggcaa caagtccacc ccatgagcgg caggtaagaa atcaatgctg agaggaccca
     2641 catgacacag agggccacca gtgccctccc acgggtgact agagctttgt aggtaggcgg
     2701 gtaacacaga cataggtagc ggtcaacagc ggttagcagc aggctgccca cagaggctgt
     2761 gaaggtcatg gtcacactgc cgatcttcag caggaagata gcgttggagt cgaccccgtg
     2821 gaagacgtgg aagatgacaa agttgcaggc gaagatcacg ctggccagga agtcagctcc
     2881 agccaagctg ctgatgaaca ggtacgaggg ctttctgcgg agccgccggg aggacaggat
     2941 aatatagagc acggccatgt tctccagggc gctcagcagc cccatcaggg tgcacagcac
     3001 cgccacggcg atctgctggc cactgctcag gatcatgtac tccttcatgg ggttgaactc
     3061 caagccaccg ttggagccgt tggtcacttc tgtctcccgg catccctcca tggcgatcgc
     3121 ggcggcagat ctcctcgcta gcataacttc gtataaagta tcctatacga agttatttgc
     3181 cttaacccag aaattatcac tgttattctt tagaatggtg caaagaataa cttcgtataa
     3241 tgtatgctat acgaagttat gaattccgct cgagataatc aacctctgga ttacaaaatt
     3301 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     3361 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     3421 tataaatcct ggttagttct tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3481 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgtttatttg tgaaatttgt
     3541 gatgctattg ctttatttgt aaccatctag ctttatttgt gaaatttgtg atgctattgc
     3601 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     3661 tatgtttcag gttcaggggg agatgtggga ggttttttaa agcggccgcg gtaaccacgt
     3721 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     3781 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     3841 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     3901 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     3961 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4021 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4081 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4141 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     4201 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4261 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     4321 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4381 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     4441 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4501 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4561 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     4621 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4681 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4741 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4801 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4861 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4921 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4981 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5041 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5101 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5161 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5221 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5281 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5341 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5401 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5461 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5521 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5581 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5641 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5701 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5761 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5821 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5881 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5941 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6001 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6061 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6121 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6181 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6241 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6301 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6361 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6421 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
